The present invention relates to a method and to a device for determining the content of L-.alpha.-aspartyl-L-phenylalanine methyl ester (Asp-PheOMe or "aspartame") in aqueous solution via enzymatically cleaving aspartame to aspartic acid and phenylalanine methyl ester, by means of a peptidase and subsequent enzymatic detection reaction. The invention further relates to an enzymatically active cell extract suitable for this purpose.
The sweetener aspartame is a dipeptide ester composed of the two L-amino acids aspartic acid and phenylalanine, which has, because of its high sweetening power and physiological acceptability, been approved in many countries within a short time and whose production is now of the order of several 1000 tons annually.
Examples of important uses for this sweetener are refreshing beverages, sweetener tablets and sweetener powders, desserts, fruit yoghurt and chewing gum.
Aspartame is completely stable only in solid form at room temperature and, in aqueous solutions, its stability depends on the time, the temperature and the pH. Possible products of hydrolytic degradation are aspartylphenylalanine, phenylalanine methyl ester, aspartic acid and phenylalanine. Hence, there is considerable interest in an analytical method for aspartame which displays maximum selectivity and is easy to implement.
Principal areas of use for such a method include process control in the production of the dipeptide and quality control for final products (foodstuffs), especially given the limited storage stability of aspartame. For example, the taste of refreshing beverages is retained only when a storage temperature of 20.degree.-25.degree. C. is maintained and the beverage is consumed within about 5 weeks. Another area of use for an analytical method for aspartame is in medicine where degradation and excretion rates have to be determined in biological samples, such as serum and urine.
It is common knowledge that aspartame analysis is usually carried out by HPLC. See, for example, J. A. Stamp & T. P. Labuza, J. Food Science 54: 1043-46 (1989). In addition, determination methods have been described which used thin-layer chromatography, capillary isotachophoresis, gas chromatography and ion-exchange chromatography, as well as amino-acid analysis. These methods demand considerable effort and are generally less suitable for high sample frequencies.
There are also already analytical methods for determining aspartame concentrations which are based on an enzymatic reaction.
In Anal. Chem. 60: 2397-99 (1988), O. Fatibello-Filho et al. describe an analytical method for determining aspartame concentration which is based on an enzymatic reaction thereof, initially with carboxypeptidase A to eliminate L-aspartic acid, and subsequent conversion with L-aspartase into fumarate and ammonia. In this case the enzymes are co-immobilized on the gas membrane of an electrode which reacts with the ammonium ions formed.
A similar method from the same research group is described by G. G. Guilbault et al., Anal. Chim. Acta 206: 369-74 (1988). This method also uses an ammonium-selective electrode on which L-aspartase is immobilized and is used to bring about direct conversion of aspartame with elimination of ammonia. In addition, the authors outline various enzymatic degradation routes, some of which take place over several stages and are said in general to take place with the evolution of ammonia. However, these methods have not been investigated in detail.
Finally, in Anal. Chim. Acta. 234: 465-69 (1990), an analytical method for aspartame is taught whereby peptidase is used to eliminate aspartic acid, which is converted in the presence of .alpha.-ketoglutarate by means of aspartate aminotransferase to L-glutamate. The assay is based on the enzymatic oxidation of the L-glutamate by glutamate oxidase and a determination of the resultant oxygen consumption.
A problem plaguing these enzymatic analytical techniques is the relatively low stability of L-aspartase. As a consequence, the information provided by a given assay is not necessarily unambiguous.